13 drought stress adaptation metabolic adjustment and regulation of gene expression

Crop plants show various adaptive and acclimatization strategies to drought stress, which range from seemingly simple morphological or physiological traits that serve as important stress

Drought stress adaptation: metabolic adjustment and regulation of gene

expression

SU J A T A BH A R G A V A1and KS H I T I J A SA W A N T

Department of Botany, University of Pune, Ganeshkhind, Pune, Maharashtra 411007, India;1Corresponding author, E-mail:

sujata@unipune.ac.in

With 2figures and 2 tables

Received March 15, 2012/Accepted August 6, 2012

Communicated by R Tuberosa

Abstract

Plants cope with drought stress by manipulating key physiological

pro-cesses like photosynthesis, respiration, water relations, antioxidant and

hormonal metabolism There exist multiple and often redundant stress

sensors, which transduce the stress signal through secondary signalling

molecules to the nucleus, where the expression of stress-response genes

is regulated Transcription factors play an important role in regulating

the expression of the stress-response genes Another level of regulation

of gene expression is at the epigenetic level and involves modi fications

either at the chromatin level or at the mRNA level Crop plants show

various adaptive and acclimatization strategies to drought stress, which

range from seemingly simple morphological or physiological traits that

serve as important stress tolerance markers to major upheavals in gene

expression in which a large number of transcription factors are induced.

Studies on contrasting crop genotypes or genetic engineering of crops

help in differentiating responses to drought from those leading to

drought tolerance Of specific importance to crop plants is not whether

they survive stress, but whether they show good yields under stress

con-ditions.

Key words: crop adaptation — drought stress responses —

stress perception— stress signalling

Global climate changes are leading to increases in temperature

and atmospheric CO2levels as well as alterations in rainfall

pat-terns Periods of inadequate rainfall leading to drought are

pre-dicted to arise more frequently under such conditions Terminal

drought conditions bring about a progressive decrease in soil

water availability to plants and cause premature plant death,

while intermittent drought conditions affect the plant growth and

development but are not usually lethal The ability to survive

longer and maintain function under intermittent or terminal

drought conditions leads to subsistence yields, which are much

lower than those observed under hydrated conditions Drought

tolerance enables plants to grow and maintain relatively high

yields in spite of drought conditions and is an outcome of the

plant’s efforts to withstand or recover from stress If the

toler-ance is restricted to that particular generation, the plant is said to

be acclimated to drought If it persists over generations, the plant

genotype is said to be adapted to drought conditions

A large number of molecular, biochemical and physiological

processes at the cellular or whole plant level are altered in

response to drought and play an important role in mitigating

stress What is crucial but difficult is to distinguish between the

responses that lead to tolerance from those that arise due to

stress-induced damage The molecular machinery involved in drought stress perception, signalling and regulation of gene expression has been fairly well understood However, there are lacunae in our understanding of how it correlates with pheno-typic alterations in the plant (Blum 2011) On the other hand, several phenotypic markers have been identified in crop plants that correlate with drought tolerance, but we know little of either the gene expression involved in these phenotypic traits or how they correlate with the yield parameters

The review attempts at going through the breadth of processes involved in giving rise to a drought-response phenotype Some

of these processes have been compared in contrasting genotypes

of crops, with the objective of understanding those that correlate with better yields under drought conditions Genetic engineering

of crop plants has also emerged as an important technique to validate the role of specific genes in giving rise to the drought phenotype

Drought Responses of Plants

Growth and water relations

A primary response of plants subjected to drought stress is growth arrest Shoot growth inhibition under drought reduces metabolic demands of the plant and mobilizes metabolites for the synthesis of protective compounds required for osmotic adjustment Root growth arrest enables the root meristem to remain functional and gives rise to rapid root growth when the stress is relieved (Hsaio and Xu 2000) Lateral root inhibition has also been seen to be an adaptive response, which leads to growth promotion of the primary root, enabling extraction of water from the lower layers of soil (Xiong et al 2006) Growth inhibition can arise due to the loss of cell turgor arising from the lack of water availability to the growing cells Water availability

to cells is low because of poor hydraulic conductance from roots

to leaves caused by stomatal closure Although a decrease in hydraulic conductance decreases the supply of nutrients to the shoot, it also prevents embolism in xylem and could constitute

an adaptive response Osmotic adjustment is another way by which plants cope with drought stress Synthesis of compatible solutes like polyols and proline under stress prevents the water loss from cells and plays an important role in turgor maintenance (Blum 2005, DaCosta and Huang 2006) Modification of growth priorities as well as reduction in the performance of photosyn-thetic organs due to stress exposure leads to alterations in carbon

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partitioning between the source and sink tissues (Roitsch 1999).

Hence, carbohydrates that contribute to growth under normal

growth conditions are now available for selective growth of roots

or for the synthesis of solutes for osmotic adjustment (Lei et al

2006, Xue et al 2008)

Photosynthesis

Water deficit–induced ABA synthesis brings about stomatal

clo-sure, which causes a decrease in intercellular carbon dioxide

concentration and inhibits photosynthesis This inhibition is

reversible and photosynthesis can resume if stomata open upon

stress removal (Chaves et al 2009) On the other hand, open

stomata and high hydraulic conductance under drought enable

photosynthesis and nutrient supply to the shoot at the cost of

risking turgor loss (Sade et al 2012) Some plants appear to

adopt the latter strategy to enable the synthesis of osmotic

metabolites from photoassimilates, which help in preventing

tur-gor loss

Carbon dioxide limitation due to prolonged stomatal closure

in the face of continued photosynthetic light reactions leads to

the accumulation of reduced photosynthetic electron transport

components, which can reduce molecular oxygen and give rise

to reactive oxygen species (ROS), thus causing indiscriminate

damage to the photosynthetic apparatus This metabolic

inhibi-tion of photosynthesis is irreversible and leads to injury (Lawlor

and Cornic 2002) Hence, photophosphorylation and ATP

gener-ation is reduced, which inhibits Rubisco activity Adaptive

responses to prevent drought-induced damage to photosynthetic

apparatus include thermal dissipation of light energy,

photode-struction of D1 protein of PSII, the xanthophyll cycle, water–

water cycle and dissociation of the light-harvesting complexes

from photosynthetic reaction centres (Niyogi 1999,

Demmig-Adams and Demmig-Adams 2006) (Table 1)

Respiration Plant growth is determined by the ratio between photosynthetic

CO2assimilation and respiratory CO2release The rate of respi-ration is regulated by processes that use the respiratory products – ATP (water and solute uptake by roots, translocation of assim-ilates to sink tissues), NADH and TCA cycle intermediates (bio-synthetic processes in growing parts of a plant), which together contribute to plant growth Under drought stress, these processes are affected and lead to a decrease in respiration rate On the other hand, increased respiratory rates have also been observed under water scarcity and these lead to an increase in the intercel-lular CO2levels in leaves (Lawlor and Tezara 2009) Higher res-piration may arise due to uncoupling of respiratory oxygen evolution from oxidative phosphorylation, which prevents the accumulation of reductants and reduces the generation of ROS Increased respiratory rates are also observed due to the activation

of energy-intensive processes like osmolyte synthesis and antiox-idant metabolism that occur under drought conditions

Interdependence of metabolic processes in chloroplasts and mitochondria has been reported (Raghavendra and Padmasree 2003) For example, mitochondria are involved in processing the glycolate produced in chloroplasts during photorespiration (Taira

et al 2004) Mitochondrial respiration also plays an important role in dissipating the NADPH generated during photosynthetic light reactions through type II NADPH dehydrogenases situated

on the matrix side (Plaxton and Podesta 2006) Hence, leaf mito-chondria act as a safety engine that enables the plant to cope with variations in chloroplast metabolism under water stress (Atkin and Macherel 2009) Plant mitochondria also prevent ROS generation within themselves by employing the alternative oxidase (AOX) pathway, in which the complexes III and IV of the respiratory electron transport system are bypassed and elec-trons are directly transferred to oxygen, with the generation of thermal energy instead of ATP (Siedow and Umbach 2000) The

Table 1: Physiological responses contributing to drought tolerance in plants

1 Adjustment of chlorophyll antenna size.

Photodestruction of D1 protein of PSII

Reduction in photosynthetic electron transport

Niyogi (1999) Vass et al (2007)

2 Thermal dissipation of light energy Uncoupling of photophosphorylation and electron

transport

Kopecky et al (2005)

3 Xanthophyll cycle, water –water cycle Protection against ROS generated in chloroplasts Demmig-Adams and Adams (2006)

Asada (1999) Jahns and Holzwarth (2012)

4 Stomatal closure, reduced hydraulic

conductance.

Delay in stomatal closure under stress

Prevention of water loss through transpiration.

Maintenance of photosynthetic activity under stress

Ghannoum (2009) Sade et al (2012)

5 Altered source –sink relations and carbon

partitioning

Induction of root growth, inhibition of shoot growth Osmolyte synthesis

Roitsch (1999) Lei et al (2006) Xue et al (2008)

6 Alternative oxidase pathway, uncoupling

proteins, NADPH dehydrogenases

Uncoupling of oxidative phosphorylation and electron transport

Xu et al (2011)

7 Prohibitins Maintenance of protein structure in inner mitochondrial

membranes

Van Aken et al (2010)

8 GABA shunt Bypass in TCA cycle, prevents the generation of

reductants

Fait et al (2007)

9 Antioxidant enzymes and substrates Scavenging ROS Miller et al (2010)

Rouhier et al (2006) Shao et al (2008)

10 Synthesis of osmotically active solutes Osmotic adjustment DaCosta and Huang (2006)

11 ABA biosynthesis Stomatal closure, regulation of aquaporin activity,

inhibition of ethylene accumulation

Thameur et al (2011) Parent et al (2009) Sharp (2002) ROS, reactive oxygen species.

AOX pathway as well as the photorespiratory pathway is

opera-tional when a plant is exposed to stress and serves a role in

maintaining cell function by preventing the accumulation of

ROS (Lambers et al 2005, Florez-Sarasa et al 2007)

In addition, the TCA cycle is modified to prevent the

genera-tion of excess reductants One of the modificagenera-tions is GABA

synthesis, in which two steps in the TCA cycle related to the

generation of reducing power are bypassed GABA accumulation

occurs during stress conditions and may constitute a stress

adap-tive response (Fait et al 2007)

Prohibitins are large protein complexes that localize to the

inner mitochondrial membrane, where they appear to play a role

in maintaining the superstructure of the inner mitochondrial

membrane and the protein complexes associated with it (Van

Aken et al 2010) They have been implied in stress tolerance

not only because of their role in protecting mitochondrial

struc-ture, but also in triggering retrograde signalling between

mito-chondria and the nucleus in response to stress, thus altering the

expression of several stress-responsive transcripts, including

AOX, heat-shock proteins (HSP) and genes involved in hormone

homoeostasis

Antioxidant metabolism

Reactive oxygen species are generated due to metabolic

pertur-bation of cells, and these cause cell damage and death While

mechanisms to prevent the generation of ROS have been

men-tioned earlier, an important adaptive mechanism consists of their

effective scavenging if and when these harmful species do arise

Antioxidant substrates like ascorbate, a-tocopherol and

carote-noids and antioxidant enzymes like superoxide dismutase,

cata-lase, ascorbate peroxidase and glutathione reductase exist in cell

organelles and the cytoplasm and play an important role in

detoxifying these reactive species (Shao et al 2008) Methionine

sulfoxide reductases are another class of antioxidant enzymes

that play a role in preventing damage to proteins due to ROS

generation in plastids (Rouhier et al 2006) These enzymes use

thioredoxin to reduce the methionine sulfoxide residues

gener-ated in proteins due to oxidative stress

Hormonal regulation

Plant hormones regulate diverse processes in plants, which

enable acclimation to stress On exposure to water deficits,

ABA synthesized in roots is known to be translocated to leaves,

where it brings about stomatal closure and inhibits plant

growth, thus enabling the plant to adapt to stress conditions

(Wilkinson and Davies 2010) In barley, fivefold increase in

endogenous ABA levels was observed in drought-tolerant

varie-ties as compared to susceptible ones, indicating its role in

improving stress tolerance (Thameur et al 2011) The role of

ABA in regulating aquaporin activity, which contributes to the

maintenance of a favourable plant water status, has also been

reported (Parent et al 2009) Improvement of shoot growth

under drought was observed when 9-cis-epoxycarotenoid

dioxy-genase (NCED3), a key enzyme in abscisic acid biosynthesis,

was overexpressed in Arabidopsis (Iuchi et al 2001) ABA

accumulation during the expression of drought tolerance is

known to bring about a reduction in ethylene production and

an inhibition of ethylene-induced senescence and abscission

ABA-deficient maize seedlings showed drought susceptibility as

well as an increase in ethylene production (Sharp 2002) Auxins

have been identified as negative regulators of drought tolerance

In wheat leaves, drought stress tolerance was accompanied by a decrease in ndole-3-acetic acid (IAA) content (Xie et al 2003) Downregulation of IAA was seen to facilitate the accumulation

of late embryogenesis-abundant (LEA) mRNA, leading to drought stress adaptation in rice (Zhang et al 2009) However, there are evidences of a transient increase in IAA content in maize leaves during the initial stages of exposure to water stress, which later drops sharply as the plant acclimates to water stress (Wang et al 2008) A rapid decline in endogenous zeatin and gibberellin (GA3) levels was also observed in maize leaves subjected to water stress, which correlated with higher levels of cell damage and plant growth inhibition Reduced cytokinin content and activity caused by either reduced biosynthesis or enhanced degradation was observed in drought-stressed plants (Pospisilova et al 2000) In alfalfa, decreased cytokinin content during drought led to accelerated senescence (Goicoechea et al 1995) Cytokinins are known to delay senescence, and an increase in the endogenous levels of cytokinins through the overexpression of the ipt gene involved in cytokinin biosynthe-sis led to stress adaptation by delaying drought-induced senes-cence (Peleg and Blumwald 2011) Cytokinins are also negative regulators of root growth and branching, and root-specific deg-radation of cytokinin contributed to primary root growth and branching induced by drought stress, hence increasing drought tolerance in Arabidopsis (Werner et al 2010)

Brassinosteroids (BRs) have also been reported to protect plants against various abiotic stresses (Kagale et al 2007) Application of BR was seen to increase water uptake and mem-brane stability, as well as to reduce ion leakage arising from membrane damage in wheat plants subjected to drought stress (Sairam 1994) However, it was shown that changes in endoge-nous BR levels did not occur during the exposure of pea plants

to water stress (Jager et al 2008)

Stress Perception and Signalling

Acclimation to stress involves processes starting from perception

of stress to the expression of large number of genes involved in the manifestation of a morphological or physiological response that increases the chances of survival under the stress condition (Fig 1)

Stress perception Molecular mechanisms that sense stress consist of a number of classes of cell surface receptors like serine/threonine-like receptor kinases called receptor-like kinases (RLKs), ion channel –linked receptors, G-protein-coupled receptors (GPCRs) and two-component histidine kinase receptors RLKs are major con-tributors to the processing of a vast array of plant developmental and environmental cues Their activity is regulated by receptor oligomerization and phosphorylation, receptor internalization and dephosphorylation or regulation at the transcriptional level (Chae

et al 2009) Brassinosteroid receptor BR1 belongs to the RLK family, which in response to BR or stress is internalized by the responding cells and the stress signal transduced Cre 1 (cytoki-nin response 1) is a two-component histidine kinase receptor that transduces signal via a phosphorelay pathway This receptor kinase, besides binding cytokinins, is also thought to act as a sensor of osmotic stress (Bartels and Sunkar 2005) Ca2+ chan-nels are responsible for the influx of Ca2+ into the cytoplasm when activated by various stress situations (Xiong et al 2002) These channels therefore act as ion channel–linked receptors of

stress GPCRs are another group of membrane receptors, which

on sensing stress activate enzymes like phospholipase C or D

which in turn release second messengers and transduce the stress

signal (Tuteja and Sopory 2008)

An intracellular receptor for ABA, PYR/RCAR, has been

shown to signal for drought stress through the activation of a

serine/threonine kinase SnRK2, in response to ABA binding

(Sheard and Zheng 2009) Because ABA synthesis is known to

be induced in response to stress, the ABA receptor can be

con-sidered to be a stress sensor

Sugar signalling has emerged as an important component of

stress responses Hexokinases were identified as glucose sensors

in plants, which played a role in repressing photosynthetic gene

expression when the hexose levels in leaf cells were high (Kim

et al 2000, Hanson and Smeekens 2009) The trehalose

biosyn-thesis pathway, in which trehalose 6 phosphate (T6P) acts as an

indicator of G6P and UDPG pool size, is known to link growth

and development to metabolite content, because both sucrose

synthesis and trehalose synthesis pathways feed into the same

metabolite pool (Vogel et al 2001, Paul et al 2008) Trehalose

phosphate phosphatases are upregulated under stress conditions

and in turn regulate the T6P levels Hence, multiple facets of

drought stress appear to be simultaneously perceived by a cell

through various receptors that respond to osmotic pressure,

membrane rigidity, metabolic status, Ca2+-level perturbations,

respectively, thereby ensuring plant response and improving the

chances of survival on drought exposure

Reactive oxygen species, which are toxic by-products of stress metabolism, also serve as important signalling molecules (Miller

et al 2010) and the oxidative signal is transduced via secondary signalling intermediates like Ca2+ or phosphatidic acid (PA)– activated serine/threonine protein kinases and mitogen-activated protein (MAP) kinases to bring about transcription of genes that play a role in acclimation (Cheeseman 2007) Due to the short half-life of ROS, redox signalling is likely to occur through the redox status of ascorbate/dehydroascorbate and reduced glutathi-one/oxidized glutathione couples (Foyer and Noctor 2000) Nitric oxide radical (NO) is synthesized in plants, probably either from arginine via a nitric oxide synthase or by nitrite reduction, and has been shown to be a component of secondary messenger cascades (Mazid et al 2011), involving cyclic GMP and Ca2+

Signal transduction Signal perception is followed by the generation of secondary signalling molecules such as protein kinases and phosphatases (serine/threonine phosphatases), phospholipids like phosphoinosi-tides (Bartels and Sunkar 2005), ROS, Ca2+, nitric oxide, cAMP and sugars, which play an important role in signal transduction (Tuteja and Sopory 2008) Many of these secondary messengers are common to diverse stress situations, indicating that cross-talk between different stress-response pathways may occur through these common signal transducers

Fig 1: Signalling cascade from perception of the drought signal to the regulation of gene expression

Mitogen-activated protein kinases bring about protein

phos-phorylation and constitute one of the major mechanisms for

sig-nal transduction They are located in the cytoplasm and consist

of three classes of enzymes (MAPK, MAPKK and MAPKKK)

that form a signalling cascade from the stress sensor located on

the plasma membrane to the regulation of gene expression in the

nucleus Translocation of the MAPK into the nucleus brings

about the activation of transcription factors through

phosphoryla-tion (Tena et al 2001)

Calcium levels in the cytoplasm have been shown to increase

transiently on stress exposure The source of this stress-induced

cytoplasmic Ca2+is either from the apoplast or from the cellular

reserves Several Ca2+ sensors like calmodulin (CaM) or

CaM-binding proteins have been identified in the cells, which

trans-duce the stress signal to the nucleus through other messengers

like phospholipase D or Ca2+-dependent protein kinases (Tuteja

and Sopory 2008)

Phospholipids like phosphoinositides that are located in the

plasma membranes are a source of several secondary signalling

molecules like phosphotidylinositol phosphates, which are

phos-phorylated by kinases (e.g PI3Kase) (Drobak and Watkins

2000) Phospholipases act on these phospholipids to generate

signalling molecules like inositol 1,4,5-trisphosphate (IP3),

diac-ylglycerol (DAG) and PA, which play a role in the transmission

of the signal across plasma membrane and in intracellular

signal-ling

Transcriptional regulation of gene expression

A large number of genes are seen to be involved in the

expres-sion of the stress phenotype (Xiong et al 2002, Shinozaki and

Yamaguchi-Shinozaki 2003) The transcriptional response

ini-tially is composed of a core set of multistress-responsive genes

and becomes increasingly stress specific as time progresses (Ma

and Bohnert 2007) DNA microarrays provide a high-throughput

means of analysing gene expression at the whole-genome level

and have been used to study the patterns of gene expression in

response to drought or high-salinity stresses in several plant

spe-cies (Seki et al 2002, Guo et al 2009, Hayano-Kanashiro et al

2009)

Some of the genes seen to be upregulated under drought stress

conditions include the genes involved in osmolyte synthesis,

genes coding for LEA proteins, aquaporins, signalling molecules

and transcription factors (TFs) Of these, the genes coding for

TFs were particularly interesting because TFs act as master

switches and trigger the simultaneous expression of a large

num-ber of stress-response genes that contribute to the stress

pheno-type (Bartels and Souer 2004) About 104 TFs, whose

expression was increased on exposure to dehydration stress, have

been identified by transcriptome analysis in Arabidopsis plants

exposed to drought stress (Rhizsky et al 2004) While most of

the transcription factors were upregulated under stress, a few

transcription factors that played a role in primary growth

pro-cesses were downregulated Drought stress–induced gene

expres-sion was seen to be regulated by TFs belonging to bZIP, AP2/

ERF, HD-ZIP, MYB, bHLH, NAC, NF-Y, EAR and ZPT2

fami-lies (Yang et al 2010) These TFs are activated at the

transcrip-tional or protein level by the transduced drought signal Because

drought stress is accompanied by an increase in ABA levels,

some TFs are activated specifically by ABA The

ABA-respon-sive TFs (ABFs) predominantly belong to the bZIP family of

TFs and bind to ABA-response elements (ABRE) present in the

promoters of stress-response genes (Jakoby et al 2002, Yoshida

et al 2010) TFs belonging to the AP2/ERF family bind to the drought-response element (DRE) present in their promoters of a large number of drought-response genes (Yamaguchi-Shinozaki and Shinozaki 2005, Maruyama et al 2009) The HD-ZIP TFs are plant specific and show the presence of a homeodomain adja-cent to leucine zipper Among several functions attributed to this family of transcription factors, one function is the regulation of ABA-dependent genes under dehydration stress (Deng et al 2002) Most of the plant MYBs consist of two repeats R2R3 (Jin and Martin 1999) and play a role in regulating the expression of dehydration-responsive genes (Abe et al 2003) The ZPT2 TFs are characterized by the presence of two zincfinger motifs sepa-rated by a single long linker These act as transcriptional repres-sors by downregulating the activity of other transcription factors (Sakamoto et al 2004) and are induced during dehydration stress

as well as with ABA treatment Transcription factors belonging

to NAC family bind to promoters of not only dehydration-response genes (Tran et al 2004), but also auxin-dehydration-response genes (Hegedus et al 2003)

The promoters of stress-response genes are known to have several types of cis elements to which TFs of the same family or different families can bind (Narusaka et al 2003, Srivastav et al 2010) Hence, gene expression under different stress situations can be combinatorially regulated by employing suitable TFs, which often form homo- or heterodimers in bringing about tran-scriptional activation under specific stress situations However, manipulations of transcription factors in engineering complex traits such as abiotic stress tolerance are known to produce unin-tended pleiotropic effects which may have adverse effects on the growth and development of plants (Abdeen et al 2010)

Post-transcriptional regulation of gene expression Besides stress-induced regulation of gene expression at the tran-scription level, stress conditions also bring about epigenetic reg-ulation of gene expression (Table 2) Stress-induced changes in histone variants, histone N-tail modifications and DNA methyla-tion have been shown to regulate stress-responsive gene expres-sion and plant development under stress Drought stress induced the expression of a variant of histone H1 called H1-S, which appeared to play a role in stomatal closure (Scippa et al 2004) ABA downregulated the expression of a histone deacetylase AtHD2C, while overexpression of this enzyme brought about enhanced expression of ABA-responsive genes and greater salt and drought tolerance than the wild-type plants (Sridha and Wu 2006) Drought-induced expression of stress-responsive genes was also seen to be associated with modifications in histones H3 and H4 Histone H3K4 trimethylation, H3K9 acetylation, H3 Ser-10 phosphorylation, H3 phosphoacetylation and H4 acetyla-tion were observed, which correlated with the expression of stress-induced genes (Sokol et al 2007) Histone acetyltransfe-rases (HATs), which interact with transcription factors, were also seen to be involved in activating stress-responsive genes Stres-ses can induce changes in gene expression through hypomethyla-tion or hypermethylahypomethyla-tion of DNA In tobacco, stress-induced DNA demethylation was observed in the coding sequence of a glycerophosphodiesterase-like protein gene, while DNA hyper-methylation was induced by drought stress in pea (Chinnusamy and Zhu 2009)

MicroRNAs (miRNAs) are ~20- to 22-nt non-coding RNAs that specifically base pair to target mRNAs and induce the cleav-age of target mRNAs or repress their translation Hence, they con-stitute a gene-silencing mechanism that regulates the expression

of target genes post-transcriptionally Regulation of

stress-response genes by miRNAs has been demonstrated recently

(Shu-kla et al 2008) For example, abiotic stress brought about

down-regulation of miR398 that targets stress-inducible Cu-Zn SOD

genes that play a role in scavenging superoxide radicals generated

in plants on exposure to stress (Sunkar et al 2006) MiRNA159

was seen to be upregulated in response to ABA, and this miRNA

silenced several MYB transcription factors that are known to

posi-tively regulate ABA responses MiR169 regulated target genes for

carbohydrate metabolism, leading to stem sugar accumulation in

sweet sorghum (Calvino et al 2011) MiRNAs miR172 and

miR395 were reported to target genes related to time offlowering

and permitted greater biomass build-up

The mRNA transcribed is processed to give rise to the mature

mRNA, and RNA-binding proteins are involved in

post-transcrip-tional RNA modifications through processes like splicing and

regulation of its stability and turnover Under stress conditions,

alternative splicing of some mRNAs coding for transcription

fac-tors has been reported in wheat (Egawa et al 2006) There are

reports indicating the occurrence of alternative splicing in at least

42% of genes in Arabidopsis during abiotic stress conditions

(Fi-lichkin et al 2010, Nakaminami et al 2012) Degradation or

sta-bilization of mRNA levels under stress conditions is brought

about by processing bodies (PBs) and stress granules (SGs),

respectively (Weber et al 2008, Xu and Chua 2011) P-bodies

are RNP complexes known to play a role in translational

repres-sion and mRNA decapping Removal of 5′ m7GDP by decapping

proteins (DCP1, DCP2) from the mRNA cap takes place in

P-bodies, which leads to further degradation of the mRNA by

exon-ucleases (XRN4) SGs have been shown to contain nuclear

pro-teins (UBP1 and RBP47), polyA+ mRNA and translation

initiation factors, which under stress conditions are observed as

distinct complexes in the cytoplasm (Weber et al 2008)

Post-translational modification of proteins also plays an

impor-tant role in the drought stress response The importance of

phos-phorylation cascades in signal transduction has already been

mentioned earlier Protein modifications are also known to affect the conformation, activity, localization and stability of transcrip-tion factors (Kline et al 2010) Ubiquitin-dependent protein deg-radation is another post-translational protein modification, which was shown to play an important role in hormonal signalling (Santner and Estelle 2009) Upregulation of an E3 ubiquitin ligase XERICO in Arabidopsis enhanced the expression of an ABA bio-synthesis gene, AtNCED3, thereby increasing the cellular ABA levels and hence drought tolerance (Ko et al 2006) In addition

to ubiquitin, plants use a variety of other polypeptide tags to post-translationally modify and regulate various intracellular proteins Small ubiquitin-like modifier (SUMO) is one such peptide that brings about sumoylation In Arabidopsis, the amount of AtSUMO1 and AtSUMO2 conjugates increased in response to various stress treatments, and when these were overexpressed, the increased sumoylation levels induced ABA-/stress-responsive genes by masking ubiquitin sites on regulatory proteins (Kurepa

et al 2003) Hence, the post-translational modifications like sumoylation and ubiquitination modulate plants response to stress

Drought Adaptation Strategies in Crop Plants

Drought-tolerant plants like xerophytes, halophytes, resurrection plants show morphological and physiological adaptations to cope with poor water availability either through growth arrest till favourable conditions return, or through shortened growth cycles comprising limited vegetative growth followed by flowering and seed set during the short periods of water availability Such adaptations are not desirable traits in crop species, which develop large yields over long growth periods Genotypes that differ in drought tolerance serve as important systems for study-ing adaptive responses to drought in crop species, and exploitation of natural variation for drought-related traits has resulted in an improvement of crop performance (Ribaut et al

2004, Reynolds and Tuberosa 2008)

Table 2: Epigenetic regulation and RNA-related processes in response to drought stress

Histone/DNA modifications

Histone variants – Chromatin state regulation H1S substitutes H1 Scippa et al (2004)

Histone modi fications – Chromatin state regulation Downregulation of HDACs

Upregulation of HATs H3K4 methylation, H3 K9 acetylation, H3S10 phosphorylation, H4 acetylation

Sokol et al (2007)

DNA methylation – Chromatin state regulation Demethylation,

Hypermethylation

Chinnusamy and Zhu (2009)

RNA-mediated regulation

miRNA-/siRNA-mediated gene silencing miR398, miR393, miR159, miR169, miR172, miR395

NATsiRNAs, tasiRNAs

Sunkar et al (2006); Shukla et al (2008); Calvino et al (2011) RNA helicase-mediated rearrangement of RNA

secondary structure

PDH45, PDH47 Owttrim (2006) RNA chaperone-mediated

RNA misfolding correction

CspA, CspB Castiglioni et al.(2008) Alternate splicing – intron retention and

generation of non-sense codons in TFs

Wdreb2 CCA1/LHY

Egawa et al (2006); Filichkin et al (2010)

RNA stress granules (SG) and processing bodies

(P-bodies) – temporary storage of mRNA

in cell, translation repression

SGs containing marker proteins eIFE4, RBP47, UBP1.

P-bodies with DCP1, DCP2, XRN4 activities

Weber et al (2008)

Xu and Chua (2011)

Post-translational regulation

Phosphorylation ABFs/AREBs, Kline et al 2010,

Sumoylation AtSUMO1, AtSUMO2 Kurepa et al 2003

HATs, histone acetyltransferases; SUMO, small ubiquitin-like modifier.

Physiological studies on contrasting genotypes provide

infor-mation on the mechanisms involved in drought tolerance and

provide a useful screening strategy for drought tolerance, albeit

at a smaller scale and often in an ‘unnatural’ drought exposure

(Fig 2) For example, drought tolerance in durum wheat was

attributed to alterations in mitochondrial metabolism The

mito-chondria showed an active AOX pathway and an uncoupling

protein, both of which played a role in the dissipation of energy

and prevented the accumulation of ROS (Pastore et al 2007) In

addition, the cytosolic NADH produced was oxidized by an

active malate/oxaloacetate shuttle in the mitochondria On

com-paring drought responses of wheat genotypes with the related

Aegilops biuncialis genotypes, a higher photosynthetic activity

was observed in Aegilops, which are adapted to drier habitats

Higher CO2 fixation was attributed to better stomatal

conduc-tance and more efficient non-radiative energy dissipation in

Aegilops (Molnar et al 2002) In comparisons made between

drought-tolerant and drought-susceptible sorghum genotypes, it

was observed that the genotypes differed in stress thresholds at

which transition from stomatal to metabolic inhibition of

photo-synthesis occurred (Bhargava and Paranjpe 2004) This has

important implications because stomatal inhibition of

photosyn-thesis is reversible and an ability to delay metabolic inhibition of

photosynthesis would facilitate the recovery from stress Tolerant

genotypes of sorghum were also seen to have higher levels of

Rubisco under drought stress than susceptible genotypes, and

this correlated with higher transcript levels of the chloroplast

chaperone HSP60, which probably protected the Rubisco protein

from drought-induced damage (Jagtap et al 1998) Source–sink

relationships also play an important role in drought tolerance of

crop plants because carbohydrate reserves are utilized for grain

filling and their availability is a critical factor in sustaining grain

filling and grain yield under drought stress (Yang and Zhang

2006) Although osmotic adjustment is another mechanism for

coping with drought stress, it is seen to be of relevance mainly

in root development into deeper soils, which can give plants

access to water This was seen in wheat lines showing better

osmotic adjustment as compared to those showing low osmotic adjustment (Morgan 1995) However, in drought-tolerant geno-types of prairie junegrass, genes involved in proline and fructan biosynthesis were seen to play an important role in drought tol-erance (Jiang et al 2010) Efficiency of antioxidant metabolism

in protecting plants against oxidative damage has been reported

in drought-tolerant crop genotypes as compared to drought-sus-ceptible ones Drought-tolerant genotypes of sorghum showed higher activities of antioxidant enzymes on exposure to stress, but not under non-stress conditions (Jagtap and Bhargava 1995)

An increase in activities of specific isozymes of antioxidant enzymes has also been reported in drought-tolerant rapeseed genotypes subjected to drought stress (Abedi and Pakniyat 2010) However, a drought-tolerant genotype Oryza longistami-nata of rice accumulated smaller amounts of ROS as well as antioxidant substrates, indicating that it had other acclimation mechanisms that prevented oxidative stress (Kumar et al 2011) The role of ABA in drought tolerance has been studied in barley genotypes differing in their ability to survive water-limiting con-ditions (Thameur et al 2011) Drought tolerance correlated with

an increase in ABA accumulation, and the genotype showing highest tolerance hadfivefold more ABA levels as compared to the susceptible genotype

At the molecular level, differences in gene expression in drought-susceptible and drought-tolerant genotypes have been observed Generally, the genes involved in protecting plants from drought stress through stress perception, signal transduc-tion, transcriptional regulatory networks in cellular responses or tolerance to dehydration were seen to be upregulated in drought-tolerant barley genotypes, while those concerned with primary metabolic processes like photosynthesis were downregulated (Guo et al 2009) In tolerant land races of maize, genes encod-ing hormones, aquaporins, HSPs, LEAs and detoxification enzymes were induced to a greater extent than in the susceptible land races (Hayano-Kanashiro et al 2009)

Many of the drought-related traits have been tagged using molecular markers, and the loci associated with these traits

Morpho-physiological Root architecture – Rice (Steele et al 2007) Anthesis-silking time – Maize (Duvick 2005) Stay-green phenotype – Sorghum (Harris et al 2007) Reversible inhibition of photosynthesis – Sorghum (Bhargava and

Paranjpe, 2004)

Mitochondrial alternative oxidase – Wheat (Pastore et al 2007) Antioxidant enzymes – Sorghum (Jagtap and Bhargava, 1995) Osmotic adjustment – Junegrass (Jiang et al 2010) ABA biosynthesis – Barley (Thameur et al 2011)

Gene regulation Signalling pathway intermediates and stress induced transcription factors – Barley (Guo et

al 2009)

Aquaporins, HSPs, LEAs – Maize

(Hayano-Kanashiro et al 2009)

Fig 2: Stress factors and stress

adaptive traits in crops

[quantitative trait loci, (QTLs)] have been used to select

geno-types that are able to yield better underfield drought conditions

For example, the ‘anthesis-silking interval’ typically increased

under water deficit and negatively correlated with yield in maize

(Duvick 2005) Screening genotypes for QTLs associated with

lower anthesis-silking interval enabled the identification of

geno-types showing better yields under water-limiting conditions In

sorghum, genotypes resistant to post-flowering drought stress,

referred to as the stay-green phenotypes, have been shown to

have a positive impact on yield under terminal drought Four

major QTLs designated as Stg2, Stg3 and Stg4 and additional

minor QTLs were identified in sorghum, which modulate the

expression of the stay-green trait (Harris et al 2007) In rice, a

QTL with a large effect on grain yield in upland rice growing

under drought stress was associated with improved root

architec-ture (Bernier et al 2007) In maize, QTLs like root-ABA and

root-yield-1.06 were identified, which were associated with root

traits, ABA concentration as well as agronomic traits, especially

grain yield across water regimes These QTLs have been used to

improve yield stability in maize under water-limiting conditions

by marker-assisted selection (Landi et al 2005, 2010) In cotton,

QTLs for a physiological trait like low osmotic potential showed

a strong association with plant height as well as with

productiv-ity in water-limiting conditions Eleven QTLs associated with

low osmotic potential were seen to be associated with thirteen

QTLs associated with seed cotton yield (Saranga et al 2004)

Similarly, two significant QTLs affecting osmotic potential

(qtlOP-2) and plant height (qtlPH-1) under drought conditions

were also identified (Saeed et al 2011) Such QTLs have been

used for developing high-yielding cotton cultivars under

water-stress conditions using marker-assisted selection In wheat, two

QTLs were found to be associated with plant height, kernel

weight and yield under varying water availability (Maccaferri

et al 2008) However, contribution of QTLs to a trait is often

low and QTLs associated with adaptive responses to drought

dif-fer across environments, while those that are constitutive are

sta-ble across environments (Collins et al 2008) Dissecting the

phenotypic traits into smaller and simpler traits, which show

high heritability in genotypes exhibiting drought tolerance, has

led to the identification of stable QTLs associated with these

traits across diverse environments (Tardieu and Tuberosa 2010)

Identification of stable QTLs enables gene discovery through

map-based cloning, and this serves as an important input in

breeding for drought tolerance using transgenic approaches Two

approaches have been mainly used for the molecular dissection

of a QTL: positional cloning and association mapping Positional

cloning enables the identification of the genetic and physical

interval cosegregating with the QTL, while association mapping

establishes a statistical association between allelic variation at a

locus and the phenotypic value of a trait across a large number

of unrelated accessions Identification of the candidate genes

associated with a QTL is difficult because a QTL is known to

span a large genomic region For example, a QTL was shown to

span a region of over 12 Mb and 310 genes in maize (Salvi and

Tuberosa 2005) A few genes identified from the QTL regions

include the CRY2 gene that is involved in cryptochrome

synthe-sis from the rice QTL forflowering time ED1, or a gene coding

for a transcription factor from the plant architecture QTL Tb1 in

maize (Salvi and Tuberosa 2005) The candidate genes or

sequences that cosegregate with the QTL are then functionally

tested with reverse genetics tools based on gene tagging,

TILL-ING or RNAi and validated for function by producing transgenic

plants (Tuberosa and Salvi 2006)

Transgenic Technology for Improved Drought Tolerance in Crops

Drought tolerance has been achieved using genetic engineering strategies to improve (i) water-use efficiency of plants, (ii) cell protection mechanisms against ROS, (iii) hormonal balance to alter the growth and development in order to avoid drought and (iv) alter the expression of drought-induced transcription factors that act as master switches in regulating a large number of downstream drought-response genes

Late embryogenesis-abundant proteins are known to accumu-late during seed desiccation and in vegetative tissues when plants experience water deficit Transgenic expression of a group 3 LEA protein from barley (HVA1) showed improved drought and salt tolerance in rice and wheat plants (Xu et al 1996, Sivamani

et al 2000) Overexpression of trehalose or polyamines was also seen to confer tolerance to abiotic stress in rice (Garg et al

2002, Capell et al 2004) Transgenic alfalfa plants overexpress-ing the antioxidant enzyme superoxide dismutase showed improved tolerance to drought stress (McKersie et al 1996) Transgenic rice plants overexpressing the isopentenyl transferase (IPT) gene, which plays a role in cytokinin biosynthesis, showed increased expression of brassinosteroid-related genes and repres-sion of jasmonate-related genes (Peleg et al 2011) Besides alterations in hormone homoeostasis, the transgenic rice plants also showed a change in source–sink relationships and a stronger sink capacity when subjected to water limitation

Attempts at overexpressing TFs that show higher expression under drought stress in tolerant as compared to susceptible geno-types (Hayano-Kanashiro et al 2009) have led to an improvement

of drought tolerance in several crops Wheat transgenics express-ing the DREB1 gene from Arabidopsis showed better tolerance to drought under glasshouse conditions (Pellegrineschi et al 2004) Rice transgenics overexpressing ABA-inducible TF (ABF3) or drought-inducible TF (DREB2) showed improved survivability and significantly higher number of panicles, respectively, in response to drought stress, as compared to wild-type plants (Oh

et al 2005, Bihani et al 2011) Overexpression of OsbZIP23 in rice exhibited significantly improved tolerance to drought and high salinity and sensitivity to ABA (Hadiarto and Tran 2011) Although transgenic technologies provide a targeted approach for improving drought tolerance, the transgenic plants are often tested under ‘unnatural’ stress conditions and it is not clear whether they would also give rise to better yields under field stress conditions However, such studies are important as they give an indication of genes that could serve as potential candi-dates for improving stress tolerance in crops, because the slow progression of dehydration that is seen in the field does not lead

to drastic changes in gene expression that are observed in potted plants (Barker et al 2005)

Climate Change and Crop Adaptation

Drought stress, especially in the tropics, is accompanied by high temperature stress, and the responses of crops to a combi-nation of these two stress factors appear to differ from the responses to either of the stresses applied singly (Sreenivasulu

et al 2007) Hence, yield responses of crop plants when exposed to abiotic stress combinations may differ from individ-ual stress exposures Besides, climate change–induced higher temperatures are predicted to increase the water requirements of crops (Nelson et al 2009) Exploiting the genetic variability available in crop species in adjusting to climate change may be

a useful strategy for identifying traits contributing to improved

tolerance to a combination of stresses expected to occur due to

climate change For example, pearl millet varieties have shown

adaptation to persistent drought as well as high temperatures in

Sahel region (Niger) of Africa Changes in morphological and

phenological characteristics (flowering time, plant height and

spike length) in varieties sampled in 2003, during which

drought and high temperatures prevailed as compared to the

same varieties sampled in 1976, when such stress situations did

not occur (Bezancon et al 2009), showed a significant shift in

adaptive traits The varieties flowered slightly earlier and had

shorter spikes in 2003 than in 1976, suggesting that selection

for these traits occurred in the face of environmental change

over this time period Two genes, PHY and PgMADS11, that

play a role in flowering time regulation were found to show

polymorphism, which could also have arisen in response to

selection In the context of climate change, a shorter life cycle

may mitigate the effect of climate change by allowing

flower-ing and seed production in stressed environments Similarly,

there would be a large number of genes involved in different

adaptive processes occurring in response to unpredictable

stres-ses arising due to climate change, which could be mined by

comparative studies on genotypes adapted to different

environ-ments

Conclusion

A number of advancements have been made in our

understand-ing of how a plant responds to drought stress Adaptation to

drought is seen to involve metabolic and morphological

altera-tions that prevent injury to plants Underlying these

physiologi-cal and morphologiphysiologi-cal alterations are molecular mechanisms that

regulate the expression of genes involved in the various adaptive

processes Although much is known now about the different type

of stress sensors, the secondary signalling molecules involved

and entire stress-specific signalling pathways have not been

deci-phered, largely due to cross-talk between different

stress-signal-ling pathways

Stress-response gene expression is regulated largely by

tran-scription factors, which in turn are subjected to very intricate

regulation at the chromatin level, RNA level and protein level

Stress-induced chromatin remodelling may mediate acclimation

responses and help a plant to cope better with subsequent stress

situations Micro-RNA-mediated gene silencing of

stress-response TFs under non-stress conditions and their activation by

downregulation of miRNA expression have emerged as another

important means of regulating downstream stress-response gene

expression

Information on the stress adaptive mechanisms shown by

drought-tolerant genotypes of crop species has been

fragmen-tary Gene expression studies in response to drought provide

information on processes involved in stress tolerance, but the

sheer magnitude of information generated in such studies

makes it a daunting task to distinguish the adaptive responses

from those that arise secondarily as an outcome of growth

arrest or cell damage Phenotypic traits associated with

drought-tolerant crops serve as important breeding tools in

identifying stress-tolerant genotypes and in introgressing the

tolerance traits into cultivated genotypes Dissecting these

com-plex phenotypic traits into simpler, heritable traits has led to

the identification of genes associated with some QTLs for

drought tolerance Understanding stress-tolerant strategies using

model plants and testing these in crop genotypes that show

adaptation to stress appear to be a useful approach in improv-ing drought tolerance of crops However for studies on adapta-tion of crop plants to complex stress situaadapta-tions arising due to climate change, there is a need to exploit the available biodi-versity in crop genotypes growing in diverse environments to understand the mechanisms involved in coping with different stress combinations

Acknowledgements

KS acknowledges University Grants Commission, Government of India, for financial assistance through award of a research fellowship.

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